Breaking and re-forming the disulfide bond at the high-potential,respiratory-type Rieske [2Fe-2S] center of thermus thermophilus: characterization of the sulfhydryl state by protein-film voltammetry

A disulfide bond, adjacent to the [2Fe-2S] cluster, is conserved in all high-potential Rieske proteins from the respiratory and photosynthetic cytochrome bc(1) and b(6)f complexes but is absent from the low-potential, bacterial dioxygenase Rieske proteins. The role of the disulfide is unclear, since cysteine mutants have resulted in only apoprotein. The high stability of the soluble Thermus thermophilus Rieske protein permits chemical reduction of the disulfide bond and characterization of the sulfhydryl (dithiol) form by protein-film voltammetry. The effect of disulfide reduction on the cluster potential is small (DeltaE(0)' 相似文献   

Reduction potentials of Rieske clusters: importance of the coupling between oxidation state and histidine protonation state

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and +150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK(a) values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pK(a), the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q(O) site of the cytochrome bc(1) and b(6)f complexes are discussed.     

The crystal structure of eosinophil cationic protein in complex with 2',5'-ADP at 2.0 A resolution reveals the details of the ribonucleolytic active site

Eosinophil cationic protein (ECP) is a component of the eosinophil granule matrix. It shows marked toxicity against helminth parasites, bacteria single-stranded RNA viruses, and host epithelial cells. Secretion of human ECP is related to eosinophil-associated allergic, asthmatic, and inflammatory diseases. ECP belongs to the pancreatic ribonuclease superfamily of proteins, and the crystal structure of ECP in the unliganded form (determined previously) exhibited a conserved RNase A fold [Boix, E., et al. (1999) Biochemistry 38, 16794-16801]. We have now determined a high-resolution (2.0 A) crystal structure of ECP in complex with adenosine 2',5'-diphosphate (2',5'-ADP) which has revealed the details of the ribonucleolytic active site. Residues Gln-14, His-15, and Lys-38 make hydrogen bond interactions with the phosphate at the P(1) site, while His-128 interacts with the purine ring at the B(2) site. A new phosphate binding site, P(-)(1), has been identified which involves Arg-34. This study is the first detailed structural analysis of the nucleotide recognition site in ECP and provides a starting point for the understanding of its substrate specificity and low catalytic efficiency compared with that of the eosinophil-derived neurotoxin (EDN), a close homologue.     

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins

Rieske proteins and Rieske ferredoxins are present in the three domains of life and are involved in a variety of cellular processes. Despite their functional diversity, these small Fe–S proteins contain a highly conserved all-β fold, which harbors a [2Fe–2S] Rieske center. We have identified a novel subtype of Rieske ferredoxins present in hyperthermophilic archaea, in which a two-cysteine conserved SKTPCX_(2–3)C motif is found at the C-terminus. We establish that in the Acidianus ambivalens representative, Rieske ferredoxin 2 (RFd2), these cysteines form a novel disulfide bond within the Rieske fold, which can be selectively broken under mild reducing conditions insufficient to reduce the [2Fe–2S] cluster or affect the secondary structure of the protein, as shown by visible circular dichroism, absorption, and attenuated total reflection Fourier transform IR spectroscopies. RFd2 presents all the EPR, visible absorption, and visible circular dichroism spectroscopic features of the [2Fe–2S] Rieske center. The cluster has a redox potential of +48 mV (25 °C and pH 7) and a pK _a of 10.1 ± 0.2. These shift to +77 mV and 8.9 ± 0.3, respectively, upon reduction of the disulfide. RFd2 has a melting temperature near the boiling point of water (T _m = 99 °C, pH 7.0), but it becomes destabilized upon disulfide reduction (ΔT _m = −9 °C, ΔC _m = −0.7 M guanidinium hydrochloride). This example illustrates how the incorporation of an additional structural element such as a disulfide bond in a highly conserved fold such as that of the Rieske domain may fine-tune the protein for a particular function or for increased stability.     

Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phosphotransfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. 15N-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored N(delta1)H tautomer of His-45, thereby rendering the N(epsilon2) imidazole unprotonated and well positioned for accepting the ATP phosphoryl group.     

Proton-coupled electron transfer at the Qo-site of the bc1 complex controls the rate of ubihydroquinone oxidation

The rate-limiting reaction of the bc(1) complex from Rhodobacter sphaeroides is transfer of the first electron from ubihydroquinone (quinol, QH(2)) to the [2Fe-2S] cluster of the Rieske iron-sulfur protein (ISP) at the Q(o)-site. Formation of the ES-complex requires participation of two substrates (S), QH(2) and ISP(ox). From the variation of rate with [S], the binding constants for both substrates involved in formation of the complex can be estimated. The configuration of the ES-complex likely involves the dissociated form of the oxidized ISP (ISP(ox)) docked at the b-interface on cyt b, in a complex in which N(epsilon) of His-161 (bovine sequence) forms a H-bond with the quinol -OH. A coupled proton and electron transfer occurs along this H-bond. This brief review discusses the information available on the nature of this reaction from kinetic, structural and mutagenesis studies. The rate is much slower than expected from the distance involved, likely because it is controlled by the low probability of finding the proton in the configuration required for electron transfer. A simplified treatment of the activation barrier is developed in terms of a probability function determined by the Br?nsted relationship, and a Marcus treatment of the electron transfer step. Incorporation of this relationship into a computer model allows exploration of the energy landscape. A set of parameters including reasonable values for activation energy, reorganization energy, distances between reactants, and driving forces, all consistent with experimental data, explains why the rate is slow, and accounts for the altered kinetics in mutant strains in which the driving force and energy profile are modified by changes in E(m) and/or pK of ISP or heme b(L).     

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu, C.-A., Yu, L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Qo) site of the bc1 complex.     

Elimination of the disulfide bridge in the Rieske iron-sulfur protein allows assembly of the [2Fe-2S] cluster into the Rieske protein but damages the ubiquinol oxidation site in the cytochrome bc1 complex

The [2Fe-2S] cluster of the Rieske iron-sulfur protein is held between two loops of the protein that are connected by a disulfide bridge. We have replaced the two cysteines that form the disulfide bridge in the Rieske protein of Saccharomyces cerevisiae with tyrosine and leucine, and tyrosine and valine, to evaluate the effects of the disulfide bridge on assembly, stability, and thermodynamic properties of the Rieske iron-sulfur cluster. EPR spectra of the Rieske proteins lacking the disulfide bridge indicate the iron-sulfur cluster is assembled in the absence of the disulfide bridge, but there are significant shifts in all g values, indicating a change in the electronic structure of the [2Fe-2S] iron-sulfur center. In addition, the midpoint potential of the iron-sulfur cluster is lowered from 265 mV in the Rieske protein from wild-type yeast to 150 mV in the protein from the C164Y/C180L mutant and to 160 mV in the protein from the C164Y/C180V mutant. Ubiquinol-cytochrome c reductase activities of the bc(1) complexes with Rieske proteins lacking the disulfide bridge are less than 1% of the activity of the bc(1) complex from wild-type yeast, even though normal amounts of the iron-sulfur protein are present as judged by Western blot analysis. These activities are lower than the 105-115 mV decrease in the midpoint potential of the Rieske iron-sulfur cluster can account for. Pre-steady-state reduction of the bc(1) complexes with menadiol indicates that quinol is not oxidized through center P but is oxidized through center N. In addition, the levels of stigmatellin and UHDBT binding are markedly diminished, while antimycin binding is unaffected, in the bc(1) complexes with Rieske proteins lacking the disulfide bridge. Taken together, these results indicate that the ubiquinol oxidation site at center P is damaged in the bc(1) complexes with Rieske proteins lacking the disulfide bridge even though the iron-sulfur cluster is assembled into the Rieske protein.     

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.     

Evidence for N coordination to Fe in the [2Fe-2S] clusters of Thermus Rieske protein and phthalate dioxygenase from Pseudomonas

Rieske-type iron/sulfur proteins and several NADH-dependent oxygenases contain Fe/S clusters with similar spectral and magnetic properties. Purified Rieske iron/sulfur protein from Thermus thermophilus contains two apparently identical [2Fe-2S] clusters in a polypeptide having only four cysteine residues, and it has been proposed that each Fe/S cluster is coordinated to two cysteine S-atoms and to an unknown number of other non-sulfur atoms (Fee, J. A., Findling, K. L., Yoshida, T., Hille, R., Tarr, G. E., Hearshen, D. O., Dunham, W. R., Day, E. P., Kent, T. A., and Munck, E. (1984) J. Biol. Chem. 259, 124-133). We have examined the Rieske protein from Thermus and the phthalate dioxygenase from Pseudomonas cepacia with electron nuclear double resonance (ENDOR) and pulsed EPR methods and report here evidence for the direct coordination of nitrogenous ligands to the Fe/S clusters in these proteins. The electron nuclear double resonance signals arising from 14N have been interpreted in terms of a strongly coupled ligand with AN = approximately 26-28 MHz and a weakly coupled ligand with AN = approximately 9 MHz. The pulsed EPR spectrum shows a rich pattern of lines in the Fourier transformed data having peaks in the range of 0.8 to 6.7 MHz. The lower frequency resonances are tentatively associated with coupling of the unpaired spin to the remote N-atoms of coordinated imidazole rings.     

The interaction of the Rieske iron-sulfur protein with occupants of the Qo-site of the bc1 complex, probed by electron spin echo envelope modulation.

The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.     

The reduction rates of DEPC-modified mutant Thermus thermophilus Rieske proteins differ when there is a negative charge proximal to the cluster

Rieske and Rieske-type proteins are electron transport proteins involved in key biological processes such as respiration, photosynthesis, and detoxification. They have a [2Fe–2S] cluster ligated by two cysteines and two histidines. A series of mutations, L135E, L135R, L135A, and Y158F, of the Rieske protein from Thermus thermophilus has been produced which probe the effects of the neighboring residues, in the second sphere, on the dynamics of cluster reduction and the reactivity of the ligating histidines. These properties were probed using titrations and modifications with diethyl pyrocarbonate (DEPC) at various pH values monitored using UV–Visible and circular dichroism spectrophotometry. These results, along with results from EPR studies, provide information on ligating histidine modification and rate of reduction of each of the mutant proteins. L135R, L135A, and Y158F react with DEPC similarly to wild type, resulting in modified protein with a reduced [2Fe–2S] cluster in <90 min, whereas L135E requires >15 h under the same conditions. Thus, the negative charge slows down the rate of reduction and provides an explanation as to why negatively charged residues are rarely, if ever, found in the equivalent position of other Rieske and Rieske-type proteins.     

A Comprehensive Phylogenetic Analysis of Rieske and Rieske-Type Iron-Sulfur Proteins

The Rieske iron-sulfur center consists of a [2Fe-2S] cluster liganded to a protein via two histidine and two cysteine residues present in conserved sequences called Rieske motifs. Two protein families possessing Rieske centers have been defined. The Rieske proteins occur as subunits in the cytochrome bc1 and cytochrome b6f complexes of prokaryotes and eukaryotes or form components of archaeal electron transport systems. The Rieske-type proteins encompass a group of bacterial oxygenases and ferredoxins. Recent studies have uncovered several new proteins containing Rieske centers, including archaeal Rieske proteins, bacterial oxygenases, bacterial ferredoxins, and, intriguingly, eukaryotic Rieske oxygenases. Since all these proteins contain a Rieske motif, they probably form a superfamily with one common ancestor. Phylogenetic analyses have, however, been generally limited to similar sequences, providing little information about relationships within the whole group of these proteins. The aim of this work is, therefore, to construct a dendrogram including representatives from all Rieske and Rieske-type protein classes in order to gain insight into their evolutionary relationships and to further define the phylogenetic niches occupied by the recently discovered proteins mentioned above.     

Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.     

Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy

The histidines in the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were labeled with (15)N, both specifically at N1' and globally, for use in heteronuclear single quantum correlation (HSQC) NMR spectroscopic analyses. The histidine-associated (15)N resonances were assigned by correlation to the C2' protons which had been assigned previously [Okar et al., Biochemistry 38, 1999, 4471-79]. Acquisition of the (1)H-(15)N HSQC from a phosphate-free sample demonstrated that the existence of His-258 in the rare N1' tautomeric state is dependent upon occupation of the phosphate binding site filled by the O2 phosphate of the substrate, fructose-2,6-bisphosphate, and subsequently, the phosphohistidine intermediate. The phosphohistidine intermediate is characterized by two hydrogen bonds involving the catalytic histidines, His-258 and His-392, which are directly observed at the N1' positions of the imidazole rings. The N1' of phospho-His-258 is protonated ((1)H chemical shift, 14.0 ppm) and hydrogen bonded to the backbone carbonyl of Gly-259. The N1' of cationic His-392 is hydrogen bonded ((1)H chemical shift, 13.5 ppm) to the phosphoryl moiety of the phosphohistidine. The existence of a protonated phospho-His-258 intermediate and the observation of a fairly strong hydrogen bond to the same phosphohistidine implies that hydrolysis of the covalent intermediate proceeds without any requirement for an "activated" water. Using the labeled histidines as probes of the catalytic site mutation of Glu-327 to alanine revealed that, in addition to its function as the proton donor to fructose-6-phosphate during formation of the transient phosphohistidine intermediate at the N3' of His-258, this residue has a significant role in maintaining the structural integrity of the catalytic site. The (1)H-(15)N HSQC data also provide clear evidence that despite being a surface residue, His-446 has a very acidic pK(a), much less than 6.0. On the basis of these observations a revised mechanism for fructose-2,6-bisphosphatase that is consistent with all of the previously published kinetic data and X-ray crystal structures is proposed. The revised mechanism accounts for the structural and kinetic consequences produced by mutation of the catalytic histidines and Glu-327. It also provides the basis for a hypothetical mechanism of bisphosphatase activation by cAMP-dependent phosphorylation of Ser-32, which is located in the N-terminal kinase domain.     

The catalytic mechanism of glucose 6-phosphate dehydrogenases: assignment and 1H NMR spectroscopy pH titration of the catalytic histidine residue in the 109 kDa Leuconostoc mesenteroides enzyme

The chemical shifts of the C(epsilon1) and C(delta2) protons of His-240 from the 109 kDa Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) were assigned by comparing 1H and 13C spectra of the wild-type and mutant G6PDs containing the His-240 to asparagine mutation (H240N). Unambiguous assignment of the His-240 1H(epsilon1) resonance was obtained from comparing 13C-1H heteronuclear multiple quantum coherence NMR spectra of wild-type and H240N G6PDs that were selectively labeled with 13C(epsilon1) histidine. The results from NOESY experiments with wild-type and H240N variants were consistent with these assignments and the three-dimensional structure of G6PD. pH titrations show that His-240 has a pK(a) of 6.4. This value is, within experimental error, identical to the value of 6.3 derived from the pH dependence of kcat [Viola, R. E. (1984) Arch. Biochem. Biophys. 228, 415-424], suggesting that the pK(a) of His-240 is unperturbed in the apoenzyme despite being part of a His-Asp catalytic dyad. The results obtained for this 109 kDa enzyme indicate that 1H NMR spectroscopy in combination with heteronuclear methods can be a useful tool for functional analysis of large proteins.     

Intermediates of the S(3) state of the oxygen-evolving complex of photosystem II

The S(3) state of the water-oxidizing complex (WOC) of photosystem II (PSII) is the last state that can be trapped before oxygen evolution occurs at the transient S(4) state. A number of EPR-detectable intermediates are associated with this critical state. The preceding paper examined mainly the decay of S(3) at cryogenic temperatures leading to the formation of a proton-deficient configuration of S(2) termed S(2)'. This second paper examines all intermediates formed by the near-IR light (NIR) excitation of the S(3) state and compares these with the light-excitation products of the S(2)' state. The rather complex set of observations is organized in a comprehensive flowchart, the central part of which is the S(3)...Q(A)(-) state. This state can be converted to various intermediates via two main pathways: (A) Excitation of S(3) by NIR light at temperatures below 77 K results presumably in the formation of an excited S(3) state, S(3), which decays via either of two pathways. Slowly at liquid helium temperatures but much faster at 77 K, S(3) decays to an EPR-silent state, denoted S(3)' ', which by raising the temperature to ca. 190 K converts to a spin configuration of the Mn cluster, characterized by g = 21, 3.7 in perpendicular and g = 23 in parallel mode EPR, denoted S(3)'. Upon further warming to 220 K, S(3)' relaxes to the untreated S(3) state. Below about 77 K and more favorably at liquid helium temperatures, an alternative pathway of S(3) decay via the metallo-radical intermediate S(2)'Z*...Q(A)(-) can be traced. This leads to the metastable state S(2)'Z...Q(A) via charge recombination. S(2)'Z* is characterized by a split-radical signal at g = 2, while all S(2)' transients are characterized by the same g = 5/2.9 (S = (7)/(2)) configuration of the Mn cluster with small modifications, reflecting an influence of the tyr Z oxidation state on the crystal-field symmetry at the Mn cluster. (B) S(2)'...Q(A) can be reached alternatively by the slow charge recombination of S(3) and Q(A)(-) at 77 K. White-light illumination of S(2)'.Q(A) below about 20 K results in charge separation, reforming the intermediate S(2)'Z*...Q(A)(-). Thermally activated branches to the main pathways are also described, e.g., at elevated temperatures tyr Z* reoxidizes S(2)' to the S(3) state. The above observations are discussed in terms of a molecular model of the S(3) state of the OEC. Main aspects of the model are the following. Intermediates, isoelectronic to S(3), are attributed to the NIR-induced translocation of the positive hole to different Mn ligands, or to tyr Z. On the basis of a comparison of the electron-donating efficiency of tyr Z and tyr D at cryogenic temperatures, it is inferred that the Mn cluster acts as the main proton acceptor from tyr Z. Water associated with the Mn cluster is assumed to be in hydrogen-bonding equilibrium with tyr Z, and an array comprising this water and adjacent water (or OH or O) ligands to Mn followed by a sequence of proton acceptors is proposed to act as an efficient proton translocation pathway. Oxidation of the tyrosine by P(680)(+) repels protons to and out from the Mn cluster. This proposed role of tyr Z in the water-splitting process is described as a proton repeller/electron abstractor.     

Iron-sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein

The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.     

Q-band ENDOR spectra of the Rieske protein from Rhodobactor capsulatus ubiquinol-cytochrome c oxidoreductase show two histidines coordinated to the [2Fe-2S] cluster.

Electron nuclear double resonance (ENDOR) experiments were performed on 14N (natural abundance) and 15N-enriched iron-sulfur Rieske protein in the ubiquinol-cytochrome c2 oxidoreductase from Rhodobactor capsulatus. The experiments proved that two distinct nitrogenous ligands, histidines, are undoubtedly ligated to the Rieske [2Fe-2S] center. The calculations of hyperfine tensors give values similar but not identical to those of the Rieske-type cluster in phthalate dioxygenase of Pseudomonas cepacia and suggest a slightly different geometry of the iron-sulfur cluster in the two proteins.